Skip to main content
Log in

Production of Extracellular Halo-alkaline Protease from a Newly Isolated Haloalkaliphilic Bacillus sp. Isolated from Seawater in Western India

  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Summary

Haloalkaliphilic, gram positive, aerobic, coccoid Bacillus sp. Po2 was isolated from a seawater sample in Gujarat, India. On the basis of 16s rRNA gene homology, Po2 was 95% related to Bacillus pseudofirmus. A substantial level of extracellular alkaline protease was produced by Po2, which corresponded with the growth and reached a maximum level (264 U/ml) during the stationary phase at 24 h. The production thereafter remained nearly static at optimal level till 36 h. Po2 could grow in the range of 0–20% NaCl (w/v) and pH 7–9, optimally at 10% NaCl (w/v) and pH 8. The protease production was salt-dependent and optimum production required 15% NaCl (w/v) and pH 8. Among the organic nitrogen sources, optimum growth and protease production (260 U/ml) were supported by the combination of peptone and yeast extract. However, growth and protease production were highly suppressed by the inorganic nitrogen sources used; with the exception of potassium nitrate, which supported both growth and protease production to limited extent (24 U/ml). Strong inhibition of enzyme production was observed at above 1% glucose (w/v). Wheat flour served as both carbon and nitrogen source supporting growth and protease production.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Alva V.A., Peyton B.M., 2003 Phenol and catechol biodegradation by the haloalkaliphile Halomonas campisalis: influence of pH and salinity Environmental Science and Technology 37: 4397–4402

    Article  CAS  Google Scholar 

  • Bakhtiar S., Andersson M., Gessesse A., Mattiasson B., Kaul R., 2003 Stability characteristics of a calcium-independent alkaline protease from Nesterenkonia sp Enzyme and Microbial Technology 32: 525–531

    Article  CAS  Google Scholar 

  • Beg K.Q., Gupta R., 2003 Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis Enzyme and Microbial Technology 32: 294–304

    Article  CAS  Google Scholar 

  • Beg K.Q., Saxena R.K., Gupta R., 2002 De-repression and subsequent induction of protease synthesis by Bacillus mojavensis under fed-batch operations Process Biochemistry 37: 1103–1109

    Article  CAS  Google Scholar 

  • Chu M.I., Lee C., & Li, S.-T., 1992 Production and degradation of alkaline protease in batch cultures of B. Subtilis ATCC 14416. Enzyme and Microbial Technology 14, 755–761

    Google Scholar 

  • Daatsellar M.C.C., Harder W., 1974 Some aspects of the regulation of the production of extracellular proteolytic enzyme by a marine bacterium Archives of Microbiology 101: 21–34

    Article  Google Scholar 

  • Denizci A.A., Kazan D., Abeln E.C., Erarslan A., 2004 Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under highly alkaline conditions Journal of Applied Microbiology 96: 320–327

    Article  CAS  Google Scholar 

  • Eichler J., 2001 Biotechnological uses of Archaeal extremozymes Biotechnology Advances 19: 261–278

    Article  CAS  Google Scholar 

  • Ellaiah P., Adinarayana K., Rajyalaxmi P., Srinivasulu B., 2003 Optimization of process parameters for alkaline protease production under solid state fermentation by alkalophilic Bacillus sp Asian Journal of Microbiology, Biotechnology and Environmental Science 5: 49–54

    CAS  Google Scholar 

  • Ferrero M.A., Castro G.R., Abate C.M., Baigori M.D., Sineriz F., 1996 Thermostable alkaline protease of Bacillus licheniformis MIR 29: isolation, production and characterization Applied Microbiology and Biotechnology 45: 327–332

    Article  CAS  Google Scholar 

  • Fujiwara N., Yamamota K., 1987 Production of alkaline protease in a low cost medium by alkalophilic Bacillus sp. properties of the enzyme Journal of Fermentation Technoogy 65: 345–348

    Article  CAS  Google Scholar 

  • Gessesse A., Kaul R.H., Gashe B.A., Mattiasson B., 2003 Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather Enzyme and Microbial Technology 32: 519–524

    Article  CAS  Google Scholar 

  • Gimenez M.I., Studdert C.A., Sanchez J., DeCastro R.E., 2000 Extracellular protease of Natrialba magadii: purification and biochemical characterization Extremophiles 4: 181–188

    Article  CAS  Google Scholar 

  • Grebeshova R., Ryshlava F., Fedorova L., Kochetova S., Babloyan O., Vinogredomna G., 1988 Using alkaline protease to intensify the processing of leather raw materiall Biotechonologiya 4: 788–791

    CAS  Google Scholar 

  • Gupta A., Roy I., Patel R.K., Singh S.P., Khare S.K., Gupta M.N., 2005 One-step purification and characterization of an alkaline protease from haloalkaliphilic Bacillus sp Journal of Chromatography A 1075: 103–108

    Article  CAS  Google Scholar 

  • Gupta R., Beg Q.K., Khan S., Chauhan B. 2002 An overview on fermentation, down stream processing and properties of microbial alkaline proteases Applied Microbiology and Biotechnology 60: 381–395

    Article  CAS  Google Scholar 

  • Hagihara, B. 1958 The Enzymes, 2nd edn. vol. 4. ed. Boyer, P. Academic Press New York

  • Heineken F.G., Connor R.J.O., 1972 Continuous culture studies on the biosynthesis of alkaline proteases, neutral proteases and alpha amylase by Bacillus subtilis NRRL-B3411 Journal of General Microbiology 73: 35–44

    CAS  Google Scholar 

  • Herbert R.A., 1992 A perspective on the biotechnological potential of extremophiles Trends in Biotechnology 10: 395–401

    Article  CAS  Google Scholar 

  • Huang Q., Yong P., Xin L., Haifeng W., Zhang Y., 2003 Purification and Characterization of an Extracellular Alkaline Serine Protease with Dehairing Function from Bacillus pumilus Current Microbiology 46: 169–173

    Article  CAS  Google Scholar 

  • Johnvesly B., Naik G.R., 2001 Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium Process Biochemistry 37: 139–144

    Article  CAS  Google Scholar 

  • Joo, H-S., Kumar, C.G., Park, G.-C., Kim K.T., Paik, S.R., Chang, C.-S. 2002 Optimization of the production of an extracellular alkaline protease from Bacillus horikoshii. Process Biochemistry 38, 155–159

    Google Scholar 

  • Joseph F., Jacques H.T., & Volker S.B., 2002 Thermophilic Protease-Producing Geobacillus from Buranga Hot Springs in Western Uganda Current Microbiology 45: 144–150

    Article  CAS  Google Scholar 

  • Kanekar P.P., Nilegaonkar S.S., Sarnaik S.S., Kelkar A.S., 2002 Optimization of protease activity of alkaliphilic bacteria isolated from an alkaline lake in India Bioresource Technology 85: 87–93

    Article  CAS  Google Scholar 

  • Kaur S., Vohra R.M., Kapoor M., Beg, k.Q., Hoondal G.S., 2001 Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2World Journal of Microbiology and Biotechnology 17: 125–129

    Article  CAS  Google Scholar 

  • Kim C., Oh M., Choi D., 1991 Production of alkaline protease by the moderate halophile Halomonas sp. ES 10Korean Agricultural Chemical Society 34: 307–311

    CAS  Google Scholar 

  • Kole M.M., Draper I., Garson D.F., 1988 Protease production by Bacillus subtilis Applied Microbiology and Biotechnology 28: 404–408

    Article  CAS  Google Scholar 

  • Madern D., Bel C., Zaccai G., 2000 Halophilic adaptation of enzymes. Extremophiles 4: 91–98

    Article  CAS  Google Scholar 

  • Malathi S., Chakraborty R., 1991 Production of alkaline protease by a new Aspergillus Flavus isolates under solid substrate fermentation conditions for use as a depilation agent Applied and Environmental Microbiology 157: 712–716

    Google Scholar 

  • Mane R., Bapat M., 2001 A study of extracellular alkaline protease from B. subtilis NCIM 2713 Indian Journal of Experimental Biology 39: 578–583

    CAS  Google Scholar 

  • Margesin R., Schinner F., 2001 Potential of halotolerant and halophilic microorganisms for biotechnology Extremophiles 5: 73–83

    Article  CAS  Google Scholar 

  • Moon S., Parulekar S., 1991 A parametric study of protease production in batch and fed batch culture of Bacillus firmus Biotechnology and Bioengineering 37: 467–483

    Article  CAS  Google Scholar 

  • Niehaus F., Bertoldo C., Ka¨hler M., Antranikian G., 1999 Extremophiles as a source of novel enzymes for industrial application Applied Microbiology and Biotechnology 5: 711–729

    Article  Google Scholar 

  • Polosina Y.Y., Zamyatkin D.F., Kostyukova A.S., Filimonov V., Fedorov O.V., 2002 Stability of Natrialba magadii NDP kinase: comparisons with other halophilic proteins Extremophiles 6: 135–142

    Article  CAS  Google Scholar 

  • Reilly T.O., Day D.F., 1983 Effect of cultural conditions on protease production by Aeromonas hydrophila Applied and Environmental Microbiology 45: 1132–1135

    Google Scholar 

  • Rozzell J.D., 1999 Commercial scale biocatalysis: myths and realities Bioorganic & Medicinal Chemistry 7: 2253–2261

    Article  CAS  Google Scholar 

  • Sanchez-Porro C., Mellado E., Bertoldo C., Antranikian G., Ventosa A., 2003 Screening and characterization of the protease CP1 produced by the moderately halophilic bacterium Pseudoalteromonas sp. strain CP76Extremophiles 7: 221–228

    CAS  Google Scholar 

  • Saeki K., Hitomi J., Okuda M., Hatada Y., Kageyama Y., Takaiwa M., Kubota H., Hagihara H., Kobayashi T., Kawai S., Ito S., 2002 A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease Extremophiles 6: 65–72

    Article  CAS  Google Scholar 

  • Singh J., Vohra R.M., Sahoo D.K., 2004 Enhanced production of alkaline proteases by Bacillus sphaericus using fed-batch culture Process Biochemistry 39: 1093–1101

    Article  CAS  Google Scholar 

  • Stan-Lotter H., Doppler E., Jarosch M., Radax C., Gruber C., Inatomi K.I., 1999 Isolation of a chymotrypsinogen B-like enzyme from the archaeon Natronomonas pharaonis and other halobacteria Extremophiles 3: 153–161

    Article  CAS  Google Scholar 

  • Stepanov V.M., Rudenskaya G.N., Revina L.P., Gryanova Y.B., Lysogorskaya E.N., Filippova I.Y., Ivanova I.I., 1992 A serine proteinase of an archebacterium, Halobacterium mediterranei Biochemical Journal 283: 281–286

    Google Scholar 

  • Studdert C.A., DeCastro R.E., Seitz K.H., Jorge J., Sanchez J.J., 1997 Detection and preliminary characterization of extracellular proteolytic activities of the haloalkalophilic archaeon Natronococcus occultu Archives of Microbiology 168: 532–535

    Article  CAS  Google Scholar 

  • Studdert C.A., Seitz M.K.H., Gilv M.I.P., Sanchez J.J., DeCastro R.E., 2001 Purification and biochemical characterization of the Haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease Journal of Basic Microbiology 6: 375–383

    Article  Google Scholar 

  • Takii Y., Kuriyama N., Suzuki Y., 1990 Alkaline serine protease production from citric acid by B. alkalohilus sub sp. Halodurans KP1239 Applied Microbiology and Biotechnology 34: 57–62

    Article  CAS  Google Scholar 

  • Uyar F., Baysal Z., 2004 Production and optimization of process parameters for alkaline protease production by a newly isolated Bacillus sp. under solid-state fermentation Process Biochemistry 39: 1893–1898

    Article  CAS  Google Scholar 

  • Xiao C., Lu J., Tian X., Li X., Zhou P., 2001 Fermentation conditions for production of alkaline elastase by alkaliphilic Bacillus XE22-4-1Wei Sheng Wu Xue Bao 41: 611–616

    CAS  Google Scholar 

  • Xu Y., Zhou P., Tian X., 1999 Characterization of two novel haloalkaliphilic archaea Natronorubrum bangense gen. nov., sp. nov. and Natronorubrum tibetense gen. nov., sp. nov International Journal of Systematic Bacteriology 49: 261–266

    Article  CAS  Google Scholar 

  • Zhang W., Xue Y., Ma Y., Zhou P., Ventosa A., Grant W.D., 2002 Salinicoccus alkaliphilus sp. nov., a novel alkaliphile and moderate halophile from Baer Soda Lake in Inner Mongolia Autonomous Region, China International Journal of Systematic and Evolutionary Microbiology 52: 789–793

    Article  CAS  Google Scholar 

  • Zhilina T.N., Detkova E.N., Rainey F.A., Osipov G.A., Lysenko A.M., Kostrikina N.A., Zavarzin G.A., 1998 Natronoincola histidinovorans gen. nov., sp. nov., A new alkaliphilic acetogenic anaerobe Current Microbiology 37: 177–185

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Financial Assistance from University Grants Commission (New Delhi, India) is acknowledged. The assistance provided by Dr B.K.C. Patel from Griffith University, Brisbane (Australia) for the 16S rRNA gene sequencing is also gratefully acknowledged. The authors are grateful to the Department of Biosciences and Saurashtra University for the necessary support.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to S.P. Singh.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Patel, R., Dodia, M., Joshi, R. et al. Production of Extracellular Halo-alkaline Protease from a Newly Isolated Haloalkaliphilic Bacillus sp. Isolated from Seawater in Western India. World J Microbiol Biotechnol 22, 375–382 (2006). https://doi.org/10.1007/s11274-005-9044-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11274-005-9044-x

Keywords

Navigation